Objective-To evaluate the ability of bovine complement to kill a variety of field isolates and laboratory strains of Brucella abortus. Design-The experimental approach was to determine the sensitivity of B abortus isolates to killing by bovine serum, and to document the role of complement in brucellacidal activity. Sample population-Six laboratory isolates and 12 field isolates of B abortus were tested. Procedure-The ability of B abortus to survive exposure to undiluted bovine serum for 2 hours at 37 C was assessed. The role of complement in killing was determined by examining the ability of heat (56 C for 60 minutes) and cobra venom factor to obliterate the activity in serum, and by detecting binding of the ninth component of bovine complement to serum-sensitive target cells. Results-Isolates of B abortus that were resistant to the bactericidal activity of normal bovine serum were revealed. These included field isolates and laboratory strains. Furthermore, the study confirmed earlier reports that bovine serum-mediated killing of B abortus is caused by the complement cascade. Conclusions-Some isolates of B abortus, like other gram-negative bacteria, were resistant to complement-mediated killing. Resistance was associated with smooth colony morphology. Isolates lacking detectable O antigen were serum sensitive.

A total of 22 clinical streptococcal isolates, predominantly Streptococcus zooepidemicus, associated with endometritis in horses were tested for their ability to withstand the natural bactericidal properties of freshly obtained blood. During a 3-hour incubation in blood from a single horse, 8 of these isolates survived and grew; the remainder were killed. To determine whether this ability to grow extended to blood of other horses, 5 of these growing isolates were tested for their ability to grow in the blood of 5 additional horses. The same 5 horses were used for each isolate. The isolates grew in blood of some of the horses, but were killed in blood of the others. However, the horse's blood that mediated killing was different for each isolate. Killing required leukocytes, but the specificity for killing appeared to reside in plasma, although plasma by itself was not bactericidal. Heat-stable and heat-labile components in plasma, interpreted as antibody and complement, respectively, appeared necessary for killing. Isolates that could grow in fresh blood lost this ability after 10 passages in artificial media. Results of these experiments of phagocytosis in fresh blood may provide helpful insights into the phagocytosis of S zooepidemicus in equine uterine f1uid.

Intraocular production of Toxoplasma gondii-specific antibody in cats has been estimated by comparing the ratio of T gondii-specific antibody in aqueous humor and serum with the ratio of total immunoglobulins in serum and aqueous humor (Goldmann-Witmer coefficient; aqueous antibody coefficient; C value). It has been proposed that in human beings, comparison of the ratio of T gondii-specific antibody in aqueous humor and serum with the ratio of antibodies against a nonocular pathogen in serum and aqueous humor is more accurate than methods using total immunoglobulin quantification. We developed an ELISA for detection of calicivirus-specific antibodies in the serum and aqueous humor of cats. By evaluating calicivirus-specific antibody concentrations in the aqueous humor of healthy and diseased cats, calicivirus was assessed as a nonintraocular pathogen. The ratio of T gondii-specific antibodies in the aqueous humor and serum and the ratio of calicivirus-specific antibodies in serum and aqueous humor were evaluated as a means of estimating intraocular T gondii-specific antibody production. A field strain of feline calicivirus was isolated, cultured, and purified. A calicivirus-specific IgG ELISA was developed for detection of feline calicivirus-specific IgG in serum and aqueous humor. Calicivirus-specific IgG was measured in the serum and aqueous humor from 3 groups of control cats. Results suggested that calicivirus is a nonintraocular pathogen in cats and that calicivirus IgG detected in aqueous humor is attributable to leakage across a damaged blood-ocular barrier. Intraocular production of T gondii-specific antibodies was estimated, using 2 formulas. The C value was calculated by multiplying the ratio of T gondii-specific IgM or IgG in aqueous humor and serum by the ratio of total immunoglobulins (using the corresponding IgM or IgG class) in serum and aqueous humor. The Ctc value (Toxoplasma-calicivirus Goldmann-Witmer coefficient) was calculated by multiplying the ratio of T gondii-specific IgM or IgG in aqueous humor and serum by the ratio of calicivirus-specific IgG in serum and aqueous humor. Serum and aqueous humor samples were obtained from 41 client-owned cats with uveitis, and T gondii-specific C values and Ctc values were calculated. Toxoplasma gondii-specific IgM or IgG C values of 10 or greater or T gondii-specific IgM or IgG Ctc values of 1 or greater were considered to be suggestive of intraocular T gondii-specific antibody production. Of the 41 cats, 20 (48.7%) had evidence of intraocular production of T gondii-specific antibody on the basis of either an IgM or IgG C value of 10 or greater. A Ctc value could not be calculated in 3 cats because calicivirus-specific IgG was not present in aqueous humor. Of the 38 cats for which Ctc values could be calculated, 25 (65.8%) had evidence of intraocular production of T gondii-specific antibody on the basis of either an IgM or IgG Ctc value of 1 or greater. The C values and Ctc values were in agreement for 75.9% of IgM containing samples and 75% of IgG containing samples. Sensitivity, specificity, predictive value of a positive test result, and predictive value of a negative test result for an IgM or IgG C value, when compared with the corresponding IgM or IgG Ctc value were determined. The results indicate that use of the C value for estimation of intraocular T gondii-specific antibody production will result in 28.6 (IgM) to 50% IgG) false-negative results and 12.5% (IgM and IgG) false-positive results, when compared with the Ctc value.

A virologic survey was conducted on calves with diarrhea associated with bovine rotavirus (BRV) on a closed dairy farm. The BRV was detected from 32 of 219 (14.6%) fecal specimens repeatedly collected from 56 calves born during the years 1992-1993, regardless of whether they had diarrhea. Most of the 32 strains were isolated from fecal specimens obtained from 2-to 6-week-old calves. After electrophoresis of doublestranded viral RNA from the 32 strains, genomic RNA migration patterns were similar to those of the predominant BRV strains isolated at the same farm during the years 1990-1991. All representative strains were identified as G serotype 6 (G6) and P type 5 (P5) by results of the virus-neutralization test and polymerase chain reaction procedure. Thus, BRV had no change in genomic RNA electropherotypes and serologic antigenicities in a closed dairy herd over a period of several years.

Toxoplasma gondii-specific IgA, IgM, and IgG were measured by ELISA in the serum and aqueous humor of 29 client-owned cats with indigenous aviates and 7 specific-pathogen-free cats tested sequentially for 20 weeks after inoculation with T. gondii. Local antibody production in aqueous humor was estimated by multiplying the aqueous humor-to-serum T gondii-specific antibody ratio by the serum-to-aqueous humor total IgG (C value) or IgG (C value) ratio. Evidence for local production of antibody in aqueous humor was defined as C value greater than 8 or CTC value greater than 1. Toxoplasma gondii-specific IgM CTC values, IgG CTC values, or IgA CTC values greater than 1 were detected in the aqueous humor of 18 of 29 (62.1%) client-owned cats with endogenous uveitis; 2 cats had IgA CTC values greater than 1 without detectable IgM or IgG in aqueous humor. Toxoplasma gondii-specific IgM was not detected in the aqueous humor of experimentally inoculated cats before or after inoculation. Immunoglobulin G C values greater than 8 were detected in all 7 experimentally inoculated cats and ranged from 10.4 to 145.5. Immunoglobulin G C values greater than 8 were first detected 4 to 8 weeks after T gondii inoculation and were undetectable by week 16 after inoculation. Immunoglobulin A C values greater than 8 were detected in 4 of 7 cats and ranged from 12.7 to 264.3. Immunoglobulin A C values greater than 8 were first detected 4 to 8 weeks after inoculation, and were detected in 2 cats during week 20 after inoculation. It was concluded that some cats infected with T gondii develop detectable concentrations of T gondii-specific IgA in aqueous humor.

Neutrophils are present in milk of cows as a means of suppressing invading pathogens during mastitis. However, the manner by which neutrophils traverse the secretory epithelia is still not clear: do they diapedese between epithelial cells or do they kill epithelial cells to gain entry into milk? We investigated the process of bovine neutrophil diapedesis across bovine mammary gland epithelium in vitro. The bovine mammary epithelial cell line MAC-T, grown on collagen-coated filters, formed a confluent monolayer with characteristic tight junctions, basal-apical polarity, and functional barriers to the dye trypan blue. Neutrophils added on the apical surface of the monolayer were stimulated to diapedese across the epithelium by the addition of Staphylococcus aureus (10(7) colony-forming units/ml) to the basal compartment. Light and transmission electron microscopy revealed the series of events for neutrophil transmigration: accumulation of neutrophils on the surface of epithelial monolayer; projection of pseudopods into intercellular junctions and movement of neutrophils between adjacent epithelial cells; and reapproximation of the lateral epithelial cell membranes and reformation of the apical tight junctions after neutrophils crossed the epithelium. Morphologically, epithelial cell damage caused by neutrophil diapedesis was not evident. This in vitro model provides a two-dimensional epithelial sheet by which neutrophil diapedesis can be qualitatively studied under defined conditions. Results of the study suggest a major mode by which bovine neutrophils diapedese across the alveolar epithelia into milk during mastitis.

Direct evidence linking alkaloids found in endophyte-infected tall fescue forage with the livestock disorder known as fescue toxicosis is lacking. Physiologic effects of fescue toxicosis include reduced serum prolactin concentration in cattle. A monoclonal antibody specific to the lysergic moiety of ergot alkaloids was developed in mice after creating an immunogen by linking lysergol to human serum albumin. The antibody was specific to the lysergic moiety and, therefore, it cross-reacted with ergot alkaloids, lysergic acid, and lysergol. The antibody did not cross-react with alkaloid derivatives that had bromated or hydrogenated lysergic ring moieties. Fescue toxicosis conditions were elicited in yearling Angus steers by permitting them to graze endophyte-infected tall fescue containing > 650 Kg/kg of ergovaline for 60 days. Passive immunization of steers by infusion of the monoclonal antibody increased serum prolactin concentration by 7 ng/ml, beginning immediately after infusion. Control steers did not respond to treatment with bovine serum albumin. Active immunization of yearling Angus heifers with immunogens containing lysergol or ergonovine linked to human serum albumin resulted in an antibody response.

Binding of endogenous and exogenous homologous IgG, and IgM to bovine neutrophils before and after in vitro migration through micropore filters, and in vivo migration through mammary tissues after intramammary injection of endotoxin was evaluated by use of flow cytometry. Immunoglobulin binding to neutrophils at 4 and 37 C was also evaluated. Before and after in vitro migration, neutrophils with endogenously bound IgG, and IgM averaged 1 and 2% and 23 and 7%, respectively. Before and after in vivo migration, IgG2 and IgM binding averaged 1 and 7% and 26 and 15%, respectively. Before and after in vitro migration, binding of purified IgG2 and IgM averaged 75 and 67% and 8 and 24%, respectively. Before and after in vivo migration, percentage of neutrophils binding purified IgG2 and IgM averaged 92 and 98% and 54 and 70%, respectively. When serum was used as a source of exogenous immunoglobulins, binding of total Igg after in vitro migration increased from 5% to 28% and of IgM from 4% to 20%. After in vivo migration, binding increased from 21% to 47% and from 24% to 56%, respectively. Exogenous binding of IgG2 at 4 and 37 C averaged 75 and 84%, and binding of IgM averaged 8% at either temperature. Endogenous IgG2 was unaffected by temperature; however, binding of IgM decreased from 23% at 4 C to 2% at 37 C. These data indicate that endogenous binding was higher for IgM before migration than after migration, in vitro and in vivo. Furthermore, migration in vivo through cellular matrices induced receptor upregulation for IgG and IgM. Source and concentration of ligand and serum components, other than immunoglobulins, appeared to contribute to receptor expression and availability. Neutrophils that were exposed to endotoxin and migrated into milk expressed more receptors than did unstimulated and nonmigrating neutrophils. The association of IgM with its receptor was temperature-dependent.

Staphylococcal antigens and immune complexes (IC) prepared from antigen and hyperimmune canine serum were tested for their effects on certain functions of mononuclear (MN) and polymorphonuclear (PMN) leukocytes (cells) obtained from healthy dogs. The effect on MN cells was studied by determining the ability of antigen or IC to augment or inhibit mitogenesis induced by phytohemagglutinin (PHA). The effect of antigen or IC on PHA cells was studied by measurement of H2O2 production as an indicator of respiratory burst. Neither the antigen nor the IC, when cultured with MN cells, was mitogenic. Coincubation of antigen or IC with MN cells and PHA resulted in a concentration-dependent decrease in mitogenesis. The decreased mitogenesis could not be overcome by addition of excess PHA, and may in part have been related to toxic effects of the antigen or IC on MN cells. When MN cells were instead preincubated with antigen or IC, then washed and stimulated with PHA, there was still a concentration-dependent inhibition of mitogenesis, although toxicity to the cells was not observed. Low concentrations of staphylococcal antigen or IC stimulated slight H2O2 production by PHA cells. When PHA cells were coincubated with IC and another stimulus (opsonized zymosan or phorbol myristate acetate), IC appeared to augment phorbol myristate acetate-, but not zymosan-induced stimulation. These results suggest that staphylococcal antigens, either alone or complexed with antibody, have the ability to stimulate PMN cells and inhibit MN cell function. Such actions may have a role in the pathogenesis of recurrent staphylococcal infection in canine patients.

Persistence of the vaccine RH strain of Toxoplasma gondii was studied by bioassay and histologically in 14 pigs. Pigs were euthanatized 2, 4, 7, 8, 14, 15, 21, 29, 36, 42, 52, 57, and 76 days after IM inoculation with 100,000 T gondii tachyzoites. Viable T gondii tachyzoites derived from the RH strain were isolated by bioassay in mice inoculated with tissues of pigs euthanatized up to 14 days after vaccination. Except for fever, pigs vaccinated IM with the RH strain remained clinically normal. Two other pigs inoculated IV with 100,000 T gondii tachyzoites of the RH strain became ill, and 1 pig was comatose by 4 days after inoculation. These findings indicate that route of inoculation may influence the response of pigs to T gondii. To evaluate protective immunity in pigs vaccinated with the RH strain, 16 age-matched pigs were allotted to 4 groups (A-D) of 4 pigs each. Eight pigs (groups A and C) were vaccinated IM with 100,000 RH strain tachyzoites and 8 pigs (groups B and D) were nonvaccinated controls. Pigs of groups A and C were challenge-inoculated orally with a lethal dose of T gondii oocysts (100,000 oocysts) 81 days after vaccination, pigs of groups B and D were inoculated similarly 220 days after vaccination. The concentration of T gondii at 3 days after challenge inoculation of pigs vaccinated 81 days earlier was reduced 100,000-fold in mesenteric lymph nodes, compared with that in a nonvaccinated pig euthanatized at 3 days after challenge inoculation. Another nonvaccinated pig became comatose and had to be euthanatized at 7 days after challenge inoculation, numerous tachyzoites were in its mesenteric lymph nodes, intestines, and liver. The vaccinated pigs generally remained clinically after challenge inoculation with oocysts. Toxoplasma gondii was not isolated by bioassays from tissues of 5 of 8 vaccinated pigs, but was recovered from all nonvaccinated pigs. Results indicate that protective immunity persisted in pigs for at least 7 months after vaccination with the nonpersistent RH strain of T gondii.